High Throughput Screening to Discover Chemical Inhibitors of Quiescin Sulfhydryl Oxidase 1 Project Summary Quiescin Sulfhydryl Oxidase 1 (QSOX1) is overexpressed in cancer cells, is active in the extracellular matrix (ECM), and is important for cancer cell proliferation and invasion. At present no specific and well-characterized chemical inhibitors exist for QSOX1. QSOX1 is an enzyme that oxides thiols during protein folding, forming disulfide bonds. Its biological role appears to be in cellular interactions with and modifications of the ECM. It is overexpressed by several malignancies including pancreatic adenocarcinoma, breast cancer, renal cell carcinoma and others. QSOX1 expression silencing using short-hairpin RNA in cancer cell lines has shown inhibition of cellular proliferation and invasion both in vitro and in vivo. These observations highlight the importance of understanding QSOX1 in carcinogenesis and as a potential therapeutic target. In this study we will use HTS and hit validation to discover novel inhibitors of QSOX1 enzymatic activity. We will scale up production of enzymatically active tumor-derived and recombinant QSOX1 protein sufficient for screening ~385,000 compounds and hit confirmation. We will perform HTS of the MLPCN libraries using a QSOX1 inhibition assay and confirm hits in the primary and orthogonal QSOX1 assays. The confirmed hits will be prioritized using the following assays: a) QSOX1, GOx and ALR oxidase panel; b) QSOX1 differential scanning fluorimetry assay; c) cellular viability, proliferation, and reactive oxygen species, d) basement membrane invasion (Boyden Chamber) assays, e) laminin incorportation into the ECM and f) mechanism of action including apoptosis, autophagy, cell-cycle assays using pancreatic and breast cancer cell lines. Select compound scaffolds will be expanded through purchase or synthesis of analogs to assess the nascent structure-activity relationship. Representatives of lead scaffolds will be scaled up and profiled using NCI60 panel and ADME/T assays in preparation for in vivo studies. In the proof-of-concept study we have performed pilot screening using the biochemical QSOX1 assay. The assay demonstrated excellent performance and was characterized with a 0.4% hit rate. Preliminary evaluation of the identified inhibitory compounds demonstrated concentration-dependent growth inhibition and cytotoxicity of MIAPaCa2 pancreatic cancer cells and two kidney cancer cell lines (RCC 786-O and UOK-117). Two of the compounds inhibited human tumor growth in nude mouse xenograft models. Our preliminary data demonstrate that each component of the testing funnel is in place and functional such that we will be able to identify chemical inhibitors of QSOX1. These inhibitors will be useful chemical probes for understanding the role of QSOX1 in normal cellular function, tumor cell biology and have promise as potential anti-neoplastic agents.